Abstract:

Topical compositions comprising an effective amount of a nitroalkene and a
carrier are used to prevent skin conditions or damage and to treat skin
conditions and damage including rosacea, eczema, psoriasis, xerosis,
dermatitis, seborrhea, thermal and radiation burns (including sunburn),
acne, alopecia, skin aging, scars, and skin inflammation.

Claims:

1. A method for the prevention of skin damage comprising: topically
applying a composition containing an effective amount of a nitroalkene in
a dermatologically acceptable non-polar carrier to skin tissue.

3. The method of claim 1 wherein the nitroalkene is present in a weight
percentage which is within one of the following
ranges:0.01%-0.025%;0.025%-0.05%;0.05%-0.10%;0.10%-0.50%;0.50%-1.0%;0.025-
%-0.50%;0.025%-1.0%;1.0%-2.0%;2.0%-5.0%;5.0%-10.00%;1.0%-5.0%;1.0%-10.0%;1-
0.0%-20.0%;20.0%-30.0%;30.0%-40.0%;40.0%-50.0%;50.0%-60.0%;60.0%-70.0%;70.-
0%-80.0%;80.0%-90.0%;90.0%-98.0%;10.0%-30.0%;20.0%-40.0%;30.0%-60.0%;40.0%-
-70.0%;50.0%-80.0%;10.0%-50.0%;10.0%-98.0%;50.0%-70.0%;50.0%-98.0%;
or70.0%-98.0%.

5. The method of claim 4, wherein the non-polar carrier comprises one or
more polyethylene glycols.

6. The method of claim 1, wherein said composition further comprises one
or more additional ingredients selected from the group consisting of:
fatty acid esters of ascorbic acid, lipoic acid, and tocotrienols and
tocotrienol derivatives and vitamin E compositions enriched with
tocotrienol or tocotrienol derivatives.

8. A method in accordance with claim 1 wherein the skin damage is skin
scarring after a wound.

9. A method for the treatment of skin damage comprising: topically
applying a composition containing an effective amount of a nitroalkene in
a dermatologically acceptable non-polar carrier to damaged skin tissue.

11. The method of claim 9, wherein the nitroalkene is present in a weight
percentage which is within one of the following
ranges:0.01%-0.025%;0.025%-0.05%;0.05%-0.10%;0.10%-0.50%;0.50%-1.0%;0.025-
%-0.50%;0.025%-1.0%;1.0%-2.0%;2.0%-5.0%;5.0%-10.00%;1.0%-5.0%;1.0%-10.0%;1-
0.0%-20.0%;20.0%-30.0%;30.0%-40.0%;40.0%-50.0%;50.0%-60.0%;60.0%-70.0%;70.-
0%-80.0%;80.0%-90.0%;90.0%-98.0%;10.0%-30.0%;20.0%-40.0%;30.0%-60.0%;40.0%-
-70.0%;50.0%-80.0%;10.0%-50.0%;10.0%-98.0%;50.0%-70.0%;50.0%-98.0%;
or70.0%-98.0%.

13. The method of claim 12, wherein the non-polar carrier comprises one or
more polyethylene glycols.

14. The method of claim 9, wherein said composition further comprises one
or more additional ingredients selected from the group consisting of:
fatty acid esters of ascorbic acid, lipoic acid, and tocotrienols and
tocotrienol derivatives and vitamin E compositions enriched with
tocotrienol or tocotrienol derivatives.

19. The method of claim 17 wherein the nitroalkene is present in a weight
percentage which is within one of the following
ranges:0.01%-0.025%;0.025%-0.05%;0.05%-0.10%;0.10%-0.50%;0.50%-1.0%;0.025-
%-0.50%;0.025%-1.0%;1.0%-2.0%;2.0%-5.0%;5.0%-10.00%;1.0%-5.0%;1.0%-10.0%;1-
0.0%-20.0%;20.0%-30.0%;30.0%-40.0%;40.0%-50.0%;50.0%-60.0%;60.0%-70.0%;70.-
0%-80.0%;80.0%-90.0%;90.0%-98.0%;10.0%-30.0%;20.0%-40.0%;30.0%-60.0%;40.0%-
-70.0%;50.0%-80.0%;10.0%-50.0%;10.0%-98.0%;50.0%-70.0%;50.0%-98.0%;
or70.0%-98.0%.

20. The method of claim 17, wherein the carrier comprises a polymer
polyether.

21. The method of claim 20, wherein the carrier further comprises a
phosphatidylcholine.

22. The method of claim 17, wherein said composition further comprises one
or more additional ingredients selected from the group consisting of:
fatty acid esters of ascorbic acid, lipoic acid, and tocotrienols and
tocotrienol derivatives and vitamin E compositions enriched with
tocotrienol or tocotrienol derivatives.

24. The method of claim 17, wherein the skin inflammation is one or more
of: acne, aging skin, alopecia, dermatitis, xerosis, eczema, rosacea,
seborrhea, and psoriasis.

25. A method for the treatment of male pattern baldness, comprising:
topically applying a composition containing an effective amount of a
nitroalkene in a dermatologically acceptable non-polar carrier to scalp
skin tissue.

[0002]The metabolism of arachidonic acid is a key element of inflammation.
In acute inflammation, there is typically a respiratory burst of
neutrophil activity that initiates cascades involving a change in the
oxidation state of the cell. Alteration in the redox state of the cell
activates transcription factors such as NFκB as well as AP1, which
then causes production of proinflammatory mediators. These mediators,
such as Tumor necrosis factorA (TFα) and various interleukins,
cause a burst of other cytokines. Arachadonic acid is released, which is
oxidized to biologically active mediators. When arachadonic acid is
oxidized via the cyclooxygenase or lipoxygenase pathways, eicosanoids
e.g. prostaglandins, leukotrines, and hyroxyeicosatetraenoic acid (HETE)
are produced, which cause erythma, edema, and free radical production.

[0003]Free fatty acids and esterified fatty acids are important components
of lipoproteins and membranes. They react with nitric oxide (.NO) and
nitric oxide derived species (NOx) to produce a variety of oxidized and
nitrated products. The oxidation of polyunsaturated fatty acids plays an
important role in biological systems and some of the metabolic products
from polyunsaturated fatty acid oxidation are important biological
mediators. The nitrated lipids produced act as signaling mediators
leading to secondary changes in protein function via electrophilic based
modifications. Nitric oxide and its metabolites also induce cyclic
guanosine monophosphate (cGMP)-independent actions in host defense
mechanisms and cell signaling. Moreover, the reaction of NOx with
eicosanoids and their impact on biosynthetic enzymes are significant
elements in the modulation of inflammatory response. Reactions of .NO and
.NO metabolites can influence catalytic reactions in eicosanoid synthesis
and modulated gene expression of related enzymes. Further, the
transcription factor NFkB mediates inducible nitric oxide synthase
expression in LPS-activated macrophages. And, .NO can serve to
down-regulate initial lipoid-mediated signaling events. Nitrated fatty
acids, particularly, nitroalkene derivatives of fatty acids, have been
detected in vivo in the blood and urine of healthy humans. (Baker et al.,
J. Biol. Chem., 2005 280:42464-42475).

[0005]Acute inflammation is often characterized generation of excited
oxygen species, e.g. superoxide anion, which damages the lipid-rich
membranes and activate the chemical mediators of the proinflammation and
inflammation cascades. These oxygenated species tend to concentrate in
hydrophobic regions. Both in or near these hydrophobic compartments, .NO
and NOx undergo a rich spectrum of reactions with oxygen species,
transition metals, thiols, lipids, and a variety of organic radicals.
These multifaceted reactions yield reactive species that transduce .NO
signaling and modulate tissue inflammatory responses.

[0006]Nitric oxide reacts with superoxide (O2.sup.-) to yield
peroxynitrite (ONOO.sup.-) and its conjugate acid, peroxynitritrous acid
(ONOOH), the latter of which undergoes homolytic scission to nitrogen
dioxide (NO2) and hydroxyl radical (OH). Also, ONOO.sup.- can react
with CO2, to form nitrosoperoxycarbonate (ONOOCO2.sup.-), which
breaks down to NO2 and carbonate (CO3.sup.-) radicals via
homolysis, or rearrangement to NO3.sup.- and CO2.

[0007]During inflammation, adaptive and protective responses are elicited
by vascular and other tissues to protect the host from its own mechanisms
directed at destroying invading pathogens. Heme oxygenase 1 (HO-1) plays
a central role in vascular inflammatory signaling and mediates a
protective response to inflammatory stresses such as atherosclerosis,
acute renal failure, vascular restenosis, transplant rejection, and
sepsis. Heme oxygenase 1 catalyzes the degradation of heme to biliverdin,
iron, and CO, the last of which has been shown to display diverse,
adaptive biological properties, including ant-inflammatory,
antiapoptotic, and vasodilatory actions. During inflammation, HO-1 gene
expression is up-regulated, with induction typically occurring
transcriptionally. Neutrophil myeloperoxidase and heme proteins such as
myoglobin and cytochrome c catalyze H2O2-- dependent oxidation
of nitrite (NO2.sup.-) to NO2, resulting in biomolecule
oxidation and nitration that is influenced by the spatial distribution of
catalytic heme proteins. These and other products are capable of
concerted oxidation, nitrosation and nitration of target molecules.

[0008]The body contains an endogenous antioxidant defense system made up
of antioxidants such as vitamins C and E, glutathione, and enzymes, e.g.,
superoxide dismutase. When metabolism increases or the body is subjected
to other stress such as infection, extreme exercise, radiation (ionizing
and non-ionizing), or chemicals, the endogenous antioxidant systems are
overwhelmed, and free radical damage takes place. Over the years, the
cell membrane continually receives damage from reactive oxygen species
and other free radicals, resulting in cross-linkage or cleavage or
proteins and lipoproteins, and oxidation of membrane lipids and
lipoproteins. Damage to the cell membrane can result in myriad changes
including loss of cell permeability, increased intercellular ionic
concentration, and decreased cellular capacity to excrete or detoxify
waste products. As the intercellular ionic concentration of potassium
increases, colloid density increases and m-RNA and protein synthesis are
hampered, resulting in decreased cellular repair. Some cells become so
dehydrated they cannot function at all.

[0009]It would be desirable to have topical treatments for rosacea,
eczema, acne, alopecia, psoriasis and inflammatory conditions in general
using compositions which disrupt the inflammatory cascades describes
above.

SUMMARY OF THE INVENTION

[0010]The present invention is directed at the selection, formulation, and
use of compounds which act with a protective response to prevent and
attenuate inflammation to provide a therapeutic effect in their control
of the pathological inflammation processes, and are also important in
providing useful biochemical tools for mechanistic investigation of the
enzymes involved.

[0011]Lipid nitration provides a means by which the proinflammatory
aspects of reactive oxygen and nitrogen species and eicosanoids are
down-regulated. The present invention is directed at the topical use of
nitroalkene compositions, including particularly, nitrolinoleic acid,
nitrooleic acid, nitrated species of arachidonic acid and nitrated
cholesteryl lineolate, as lipid signaling mediators to reduce
inflammation and inflammation mediated skin conditions.

[0012]It is an object of the invention to provide therapeutically
effective topical compositions of nitroalkene and carrier to prevent,
treat, or otherwise improve the skin conditions through topical
application.

[0013]It is an object of the invention to provide methods for preventing
and/or treating skin damage that comprise applying a composition
containing nitroalkene in a dermatologically acceptable carrier to skin.

[0014]In accordance with the present invention, topical methods of use of
nitroalkenes to prevent or treat rosacea, eczema, psoriasis, xerosis,
dermatitis, seborrhea, acne, alopecia, other types of skin inflammation,
skin aging, and scarring are disclosed.

[0015]The amount of nitroalkene necessary to treat skin or prevent skin
damage is not fixed per se and is necessarily dependent upon the amount
and identity of any adjunct ingredients in the preparation. In some
typical embodiments of the invention, the composition comprises about
0.025% to about 70% by weight nitroalkene in a dermatologically
acceptable polymer polyether and/or phosphatidycholine carrier.
Optionally, at least one or a mixture of lipoic acid, fatty acid ester of
ascorbic acid may be added to the composition.

[0016]In some typical embodiments of the invention, the method for
preventing and/or treating skin damage comprises applying a composition
containing about 0.025% to about 70% by weight of nitroalkene in a
dermatologically acceptable carrier. Optionally, at least one or a
mixture of lipoic acid or fatty acid ester of ascorbic acid may be added
to the composition.

DETAILED DESCRIPTION OF THE INVENTION

[0017]U.S. Pat. No. 6,924,309 to Ferrante et al., suggests that hydroxyl,
hydroperoxy, epoxy and peroxy substituted nitro compounds may be useful
due to their ability to inhibit IFN-γ. Such compounds are alkenes,
typically containing 3 or more double bonds which must be formed through
synthetic reactions such as the Michael addition.

[0018]Nitrated fatty acids serve as mediators of physiological and
pathophysiological cell signaling processes. Functional consequences of
these signaling mechanisms have been shown in inhibition of platelet and
neutrophil functions, activation of the transcription factor Nrf2 which
upregulates gene expressions of cytoprotective phase 2 protienases such
as heme oxygenase-1 (HO-1), inhibition of LPS-induced cytokine release in
moncytes, increased insulin sensitivity and glucose uptake in adipocytes,
and relaxation of preconstricted rat aortic segments.

[0019]The mechanism of .NO release by nitrated fatty acids is not fully
understood. Modified Nef reaction mechanisms (Schopfer et al., J. Biol.
Chem. 2005 280:19289-97) and isomerization of the nitro-fatty acid to the
correseponding nitrite derivative through a hemolytic scission of the
--NO2 group have been proposed (Lima et al., Free Radic. Biol. Med.
2005 39:532-39). Another proposal is that upon administration in vivo,
nitro fatty acids undergo reversible and exchangeable electrophilic
reactions with nucleophilic targets and are metabolized predominantly via
saturation of the double bond and beta-oxidation reactions that terminate
at the site of acyl-chain nitration (Rudolph et al., J. Biol Chem, 2009
284:1461-73). Reversible nitroalkylation reactions with glutathione (GSH)
and the Cys and His residues of proteins, demonstrate the electrophilic
nature of the β-carbon adjacent to the nitro-bonded carbon. Nitrated
fatty acids have been reported as potential endogenous ligands for
PPARγ because of their ability to react with cellular nucleophiles
to postranslationally modify protein structure, function, and
localization (Baker et al., Free Radic Biol Med, 2009 46:989-1003).

[0021]In 2004, Baker et al. reported the isolation of two positional
isomers of nitro-linoleic acid (LNO2) that were found in red blood
cell membranes and plasma (Proc. Natl. Acad. Sci. U.S.A. 2004
101:11577-82). Since then, LNO2 has been shown to inhibit vascular
smooth muscle cell proliferation by activating the nuclear
factor-erythroid 2-related factor 2 (Nrf2) (Villacorta et al., Am J
Physiol Heart Circ Physiol, 2007 293:H770-6). Nrf2 is a transcription
factor that is in the inactive form at the cytosol due to the inhibitory
activity of Keap1. Keap1 is highly reactive to nitroalkylation since it
constitutes a cysteine-rich protein. When activated, Nrf2 migrates to the
nucleus and binds as a heterodymer to the antioxidant response element
(ARE) in DNA, activating the expression of cytoprotective phase 2 enzymes
i.e. heme oxygenase 1 (HO-1), superoxide dismutase, catalase, glutathione
peroxidases, the peroxy redoxines, NADPH, and quinone oxyreductases.

[0022]LNO2 also inhibits fMLP and PMA-mediated active of human
neutrophils and blocks NF-kB activity, inhibits Keap1, resulting in
activation of Nrf2 which induces expression of cytoprotective molecules.
Moreover, LNO2 and OANO2 have been shown to exert cell
signaling action via ligation and activation of PPARγ. PPARγ
activation can effect modulation of metabolic and cellular
differentiation genes and regulation of inflammatory responses. In the
vasculature, PPARγ is expressed in monocytes, macrophages, smooth
muscle cells, and endothelium and plays a central role in regulating the
genes related to lipid trafficking, cell proliferation and inflammatory
signaling. The removal of LNO2-GSH adducts by MRP1 shows that
electrophilic reactivity likely plays a role in inhibiting LNO2
dependent PPARγ transcription.

[0024]The use of nitroalkenes in topical applications for improvement of
skin conditions has not been described in the literature. The present
invention comprises topical nitroalkene treatments which improve skin
condition by disrupting the cascade of reactions that cause inflammation.

[0025]Nitroalkenes consist of the general formula NO2-A-B, in which A
is a saturated hydrocarbon chain and B is (CH2)n(COOH)m in
which n is 0 to 2 and m is 0 to 2; and the derivatives thereof having
further one or more substitution selected from the group consisting of
hydroxyl, hydroperoxy, epoxy and perxoy. In a preferred embodiment of the
invention, A is a hydrocarbon chain of 17 atoms and B is CH2(COOH).
More specifically, the preferred compounds are nitro-linoleic acid,
nitro-oleic acid, nitrated arachidonic acid, or nitrated cholesteryl
lineolate. Of these, nitro-linoleic acid, and nitro-oleic acid are
preferred.

[0026]Additional nitroalkene compounds that may be used in accordance with
the invention include the compounds disclosed in Ferrante, U.S. Pat.
No.6,924,309; and Freeman, U.S. Patent Publication No. US 2007/0232579
A1, the disclosures of which are hereby incorporated by reference, those
discussed by Trostchansky and Rubbo, Free Radical Biology & Medicine 44
(2008) 1887-96; and Baker et al., Free Radical Biology & Medicine, 46
(2009) 989-1003, the disclosures of which are incorporated herein by
reference.

[0027]The most preferred compounds are those in which a NO2 group is
located adjacent a double bond in the carbon chain, such as in the
compounds illustrated in Table 1 below.

[0028]Polyunsaturated nitro compounds have been compared to fatty acids,
which have a variety of biological activities including anti-inflammatory
properties, since the nitro group is chemically similar to COOH groups of
essential fatty acids with regard to size, charge and shape. In addition,
the nitro compounds are a group of relatively stable compounds and are
resistant to β-oxidation by preventing CoA thiosester production,
which is the first step in β-oxidation of fatty acids. Because of
this, they are not readily incorporated into lipids and are more likely
to be present in a free form. Polyunsaturated nitro compounds have the
ability to penetrate cells and tissues suggesting their use to prevent
oxidative damage including anti-aging agents. Moreover, their ability to
inhibit interferon-gamma (IFN-γ) (a cytokine of Th-1 cells) makes
the substances useful in the treatment of allergy and skin diseases where
IFN-γ plays a pathogenic role e.g. atopic dermatitis.

[0029]Synthesis and Formation

There are various experimental approaches to chemically synthesize
nitrated unsaturated fatty acids. Preparation of nitroalkenes of the
present invention may be possible through any of the routes disclosed in
U.S. Pat. No. 6,924,309, and in Trostchansky and Rubbo supra.

[0030]Lipid nitration in vivo may also arise through one or more of
several different pathways, namely: 1) nitrogen dioxide radical reacts
with unsaturated lipids and lipid radicals leading to isomerized,
oxidized and/or nitro-allylic, nitroalkane, dinitro, or nitro-hydroxy
lipid derivatives; 2) peroxynitrite and peroxynitrous acid homolyze
yielding nitrogen dioxide radical and hydroxyl radical which mediate
oxidation, nitrosation, and nitration reactions; 3) addition of nitronium
ion by electrophilic substitution at the double bond; 4) reaction of a
carbon-centered radical with nitrogen dioxide radical both coming from a
caged radical rearrangement of unstable alkyl peroxynitrite
intermediates; and 5) nitroaldol addition by combining known precursors
yielding a nitro-alcohol product i.e. activation of hydroxyl group
followed by dehydration reaction via catalytic base.

[0031]Compositions

Only effective amounts of topical compositions containing nitroalkene are
needed to achieve the intended benefits including prevention and
treatment of inflammatory skin conditions, aging and scarring. By
"effective amount" is meant an amount of active ingredient(s) sufficient
to turn on the Nrf2 transcription factor and inhibit NF-kB and/or
upregulate expression of other protective ligands, thereby inhibiting the
products of the arachidonic acid cascade which leads to the activation of
transcription factors that direct the cell nucleus in producing
pro-inflammatory cytokines.

[0032]The topical compositions are based on a carrier in which the
nitroalkene is soluble per se or is effectively solublized (e.g. as an
emulsion or microemulsion). The carrier is dermatologically acceptable in
the sense of not bringing about any adverse effect on the skin areas to
which it is applied. The carrier preferably is appropriately selected for
topical application, and forms a film or layer on the skin to which it is
applied so as to localize the application. The nitroalkene is applied in
admixture with the dermatologically acceptable carrier or vehicle (e.g.
as a lotion, cream, gel, ointment, soap, stick, or the like) to as to
facilitate topical application and provide therapeutic effects.

[0034]A phosphatidycholine based carrier is another possible embodiment.
Phosphatidylcholine, commonly called lecithin, is a mixture of
diglycerides of stearic, palmitic, and oleic acids, linked to the choline
ester of phosphoric acid. It can be isolated from eggs, soybeans, and
other biological materials, chemically synthesized, or obtained
commercially from many sources. Carrier formulations as disclosed in U.S.
Pat. No. 7,182,956, the disclosure of which is hereby incorporated by
reference, including polyenylphosphatidycholine enriched
phosphatidycholine and polyglycol mixtures, are particularly preferred.

[0035]The quantity of the nitroalkene active ingredient in the carrier may
be varied or adjusted widely depending upon the particular application,
the potency of the particular compound, and the desired concentration.
Generally, the quantity of nitroalkene active ingredient will range
between 0.025% to 70% by weight of the topical composition. Generally,
lower concentrations of nitroalkene active ingredients in a carrier are
suitable, depending upon the application regimen and the active and
adjunct ingredients employed.

[0036]The following weight percentage of nitrolalkene ranges are expected
to be useful for different applications. These weight percentage ranges
are applicable particularly to LNO2 and OANO2.

[0068]The topical composition of the invention can contain additional
ingredients commonly found in skin care compositions and cosmetics, such
as, for example, tinting agents, emollients, skin conditioning agents,
emulsifying agents, humectants, preservatives, antioxidants, perfumes,
chelating agents, etc., provided that they are physically and chemically
compatible with other components of the composition.

[0069]As nitroalkenes are very reactive molecules, a nitroalkene topical
composition desirably includes a substantial antioxidant and preservative
system. In one preferred embodiment, the antioxidant system is Oxynex®
AP, Oynex® LM, or Oxynex® K. The preferred embodiments uses fatty
acids of Vitamin C, specifically ascorbyl palmitate, as a significant
component of the antioxidant system. Antioxidants are typically present
in an amount ranging from about 0.025% to about 5.00% by weight of the
composition, include, but are not limited to, butylated hydroxy toluene
(BHT); vitamin C and/or vitamin C derivatives, such as fatty acid esters
of ascorbic acid, particularly asocorbyl palmitate; butylated
hydroanisole (BHA); phenyl-α-naphthylamine; hydroquinone; propyl
gallate; nordihydroquiaretic acid; vitamin E and/or derivatives of
vitamin E, including tocotrienol and/or tocotrienol derivatives; calcium
pantothenates; green tea extracts; mixed polyphenols; and mixtures of any
of these. As mentioned above, particularly preferred antioxidants are
those that provide additional benefits to the skin such as ascorbyl
palmitate. Preservatives are typically present in an amount ranging from
about 0.5% to about 2.0% by weight percent, based on the total
composition.

[0070]Emollients, typically present in amounts ranging from about 0.01% to
5% of the total composition include, but are not limited to, fatty
esters, fatty alcohols, mineral oils, polyether siloxane copolymers, and
mixtures thereof. Humectants may be present in amounts ranging from about
0.1% to about 5% by weight of the total composition. Non-polar humectants
are preferred. Emulsifiers, typically present in amounts from about 1% to
about 10% by weight of the composition, include, but are not limited to,
stearic acid, cetyl alcohol, stearyl alcohol, steareth 2, steareth 20,
acrylates/C10-30 alkyl acrylate crosspolymers, and mixtures thereof.
Chelating agents, typically present in amounts ranging from about 0.01%
to about 2% by weight, include, but are not limited to, ethylenediamine
tetraacetic acid (EDTA) and derivatives and salts thereof, dihydroxyethyl
glycine, tartaric acid, and mixtures thereof.

[0071]Some embodiments of this invention contain at least one other
adjunct ingredient in addition to nitroalkene(s). Fat-soluble fatty acid
esters of ascorbic acid (vitamin C) are employed as an adjunct ingredient
as well as an antioxidant in some embodiments. The more
oxidation-resistant saturated fatty acid esters of ascorbic acid are
preferred, including, but not limited to, ascorbyl laurate, ascorbyl
myristate, ascorbyl palmitate, ascorbyl stearate, and ascorbyl behenate.
Ascorbyl palmitate is used in one prefrerred embodiment. Other possible
adjunct ingredients include, but are not limited to one or more of: amino
acids, lipoic acid; or tocotrienols and tocotrienol derivatives and
vitamin E compositions enriched with tocotrienols or tocotrienol
derivatives

Generally in the practice of the methods of the invention, the topical
composition is topically applied to the skin areas, such as that of the
face, at predetermined intervals with gradual improvement in the skin
areas expected with each successive application.

[0080]Topical compositions containing nitroalkene according to the present
invention can be topically applied to and absorbed by the skin tissue.
Nitroalkenes activate Nrf2, PPARγ, modify NF-kB gene expression or
inhibit IFN-γ and TNF and human neutrophils and macrophage
degranulation as well as cytokine release, thus preventing the cascade of
reactions that lead to inflammation and degranulation.

[0081]While not wishing to be bound by any theory, it is possible that
nitroalkenes react with cellular nucleophiles to postranslationally
modify protein structure thus affecting XOR, GAPDH or any of the
aforementioned inflammatory regulating compounds. Another possibility is
that nitro compounds of the present invention work in a similar manner to
the polyunsaturated nitro compounds of Ferrante. Moreover, nitroalkenes
of the invention may enhance Nrf2 nuclear translocation, activate
PPARγ, or modify the NF-kB subunit 65 that encodes proinflammatory
cytokines.

[0082]Nitroalkenes undergo salvation in aqueous solutions and therefore
tend to decay faster in phosphate buffer than in organic solvent. Release
of .NO is also observed in aqueous environments. The release is
independent of the presence of thiol adjuvants such as cysteines. The
capacity to release .NO is has been related to the vasorelaxing
properties of nitroalkenes observed in rat aortic ring, specifically
AANO2 and AA(OH)NO2. The stability of nitro-fatty acids is
better in hydrophobic environments. The release of .NO from LNO2 is
inhibited when inserted in phosphatidylcholine/cholesterol liposomes and
it is considered stable in hydrophobic environments.

[0083]Methods and compositions of the present invention are expected to be
particularly useful for treating skin tissue suffering from or damaged by
inflammatory conditions. The methods and compositions are expected to be
useful in prevention and treatment of the following conditions: rosacea,
eczema, psoriasis, xerosis, dermatitis (both contact dermatitis and
atopic dermatitis), seborrhea, thermal and radiation burns (including
sunburn), acne, alopecia, aging-induced skin tissue degeneration, scars,
and other types of skin inflammation.

[0084]Skin aging bears some similarities to chronic inflammatory
conditions. Cell aging is due in part to free radical damage, which takes
place mostly within the cell membrane. The cell membrane is most
susceptible to attack by free radicals because of its dense molecular
structure largely comprising lipids and lipoproteins, which are easily
oxidized by reactive oxygen species. In skin, reactive oxygen species
such as singlet oxygen, the superoxide anion, and hydroxyl radicals, as
well as other free radicals, are generated in normal metabolism, as well
as through ultraviolet sun exposure, other forms of radiation, other
environmental factors such as pollution or exposure to chemicals in the
home or workplace, and the like, active in the arachidonic acid cascade.
As in inflammation, free radicals activate chemical mediators that
increase phospholipidase A2 resulting in the release of arachidonic acid
from the cell membrane which is then oxidized by lipooxygenase and
cyclooxygenase enzymes which produce leukotrines and prostaglandins,
stimulating the inflammation cascade. It is expected that the topical
application of nitroalkene compounds according to the invention will be
effective to protect collagen and elastin from degradation by matrix
metallopritenases. After treatment for a period of time, it is expected
that elasticity and a supple feeling will return to the skin, fine lines
and wrinkles will be reduced, and skin coloring will even out. The
present invention thus includes use of nitroalkene compositions to
prevent and treats skin aging, as well as both preventing and treating
skin damage.

[0085]Skin inflammation appears in many conditions of alopecia, inclusing
male pattern baldness. I believe that the compositions and methods of the
present invention will also be effective to prevent or treat alopecia by
regular application of nitroalkene compositions.

[0086]Another expected use of the compositions of the present invention is
in encouraging wound healing without scarring, and also, in remodeling
scarred skin to a smoother, unscarred appearance. Scars result from wound
healing, which occurs in three separate phases: inflammation, formation
of granulation tissue, and matrix formation. (See Plast. Reconst.
Surgery, 2008 122:1068-78; incorporated herein by reference). During the
first phase, damage to endothelial cells, complement, and platelets at
the wound site release chemotactic factors that result in the infusion of
neutrophils, lymphocytes and macrophages, which aids in the removal of
infection and foreign debris. As in all inflammatory processes, there is
generation of free radicals, which damages cell membranes and results in
formation of oxidized proteins and fats, and cross-linked new collagen,
laying a scaffold for the next phase. At the end of the inflammatory
phase, the granulation phase begins with an influx of fibroblasts and
endothelial cells to the wound. Other key cells in this phase are
macrophages and platelets. Macrophages induce the beginning of
granulation by releasing platelet-derived growth factor (PDGF), tumor
necrosis growth factor (TGF)-α, and an epidermal growth factor-like
substance. Activated platelets release epidermal growth factor (EGF),
PDGF, TGF-α, and TGF-β. Together these play roles in the
re-epithelialization process wherein keratinocytes cells migrate in
sheaths over a provisional matrix consisting primarily of fibrin,
fibronectin, type V collagen, and tenascin, and produce their own
fibronectin receptors. Once re-epithelilization has occurred,
keratinocytes resume their normal differentiated form, and matrix
formation begins. Matrix formation consists primarily of the construction
of dermal matrix, which is regulated by fibroblasts. Chemotaxis of
fibroblasts results in the production of abundant quantities of
hyaluronate, fibronectin, and types I and III collagen. These components
comprise the bulk of the provisional extracellular matrix in the early
part of this wound repair phase. Hyaluronic acid (HA) creates an
open-weave pattern in the collagen/fibronectin scaffold, facilitating
fibroblast movement. HA production falls after about the fifth day of
wound healing, and levels of chronroitin sulfate in dermatan sulfate
increase. Fibronectin deposits in the collagen, and wound contraction
begins. Biochemically during the contraction stage, hyaluronidase and
proteinase are present, type I collagen synthesis is stimulated, and
increased levels of chronroitin sulfate, dermatin sulfate and
proteoglycans are observed; together these restructure the matrix. At the
end of the healing process, the final scar shows collagen fibers mostly
parallel to the epidermis.

[0087]Hypertrophic and keloid-type scars result in extension of scar
tissue so that a bulky lesion results. A keloid is an exuberant scar that
proliferates beyond the original wound. It should be noted that keloids
only occur in humans, often causing burning, stinging and itching
sensations as well as cosmetic embarrassment. The etiology of unsightly
keloid formation is not known. However, in keloids, fibronectin formation
continues for years, while fibronectin formation in normal scars
disappears within a few days after wound closure. Keloid scars exhibit a
high rate of collagen synthesis in comparison to normal scars, and a low
proportion of cross-linked collagen. Hypertrophic scars sometimes are
difficult to distinguish from keloid scars histologically and
biochemically, but unlike keloids, hypertropic scars remain confined to
the injury site and often mature and flatten out over time. Both types
secrete larger amounts of collagen than normal scars, but typically the
hypertrophic type exhibits declining collagen synthesis after about six
months. However, hypertrophic scars contain nearly twice as much
glycosaminoglycan as normal scars, and this and enhanced synthetic and
enzymatic activity result in significant alterations in the matrix which
affects the mechanical properties of the scars, including decreased
extensibility that makes them feel firm.

[0088]Atrophic scars are characterized by a thinning and diminished
elasticity of the skin due to a loss of normal skin architecture. An
example of an atrophic scar is striae distensae, also known as stretch
marks. Striae commonly occur in postpartum women after childbirth and
also during times of larger-than-average weight gain and also in
association with steroids. Atrophic scars are sometimes also observed
after trauma, infection and disease, and may show loss of surface
markings and smoothness or dry, fine wrinkles over time.

[0089]Formation of scars, especially hypertrophic and keloid scars, is
dependent on systemic growth factors such as interleukins and other
cytokines, and their influence on fibronectin and collagen biossynthesis.
Cytokines are released and are present in the wound healing process and,
as mentioned above, are released in the inflammatory stage. Growth
factors and other cytokines vary in the inflammatory stage and are
released in amounts based, among other complex interactions, upon the
redox state of the cells. The presence of free radicals in the
inflammatory stage plays an important factor in wound healing. Factors
that increase the presence of free radicals, such as infection,
radiation, and continued trauma, may instigate hypertrophic and keloid
scar formation. It is important to note that cytokines have been
suggested to regulate nitric oxide synthetase, which controls the
formation of nictric oxide, which plays an important role in signal
transduction in the cells. It is also known that nitric oxide synthetase
activity is aberrant in keloid scars when compared to normal tissue (Lim,
T. C., et al., Plastic and Reconst. Surgery, 1996, 98: 911-912).
Hypertrophic and keloid scars also show inflammatory activity that is not
seen in mature scars.

[0090]The nitroalkene compositions and methods of the present invention
are expected to be effective to reduce scarring during the process of
wound healing and to remodel previously damaged or scarred skin. After
treatment for a period of time, decreased inflammation, irritation, and
erythema of the skin should occur, with a flattening of the scars and
evening out of skin coloring.

[0091]Acne is the most common pustular condition of the skin, disfiguring
afflicted persons with inflammatory and noninflammatory lesions
(including pustules, papules and comedones) during the active phase, and
with atrophic scars afterwards. It occurs most commonly in teenagers, but
is not confined to adolescents. A significant number of persons continue
to seek advice on treatment for acne after the teenage years (Collier et
al., J. Am. Acad. Dermatology, 2008 58:56-59). Although acne is generally
considered to be self-limiting, its social effects can be substantial,
and it may have its most severe effects on the psyche (Am. J. Clinical
Dermatology, 2008 9(5):279-284). In about 60% of teenagers, disease
severity and embarrassment are sufficient for them to self-medicate with
proprietary preparations and/or seek medical advice.

[0092]Acne is a multifactorial disease affecting the pilosebaceous units
of the skin. Each unit consists of a large, multilobed sebaceous gland, a
rudimentary hair and a wide follicular canal lined with stratefied
squamous epithelium. They are found over most of the body surface but are
largest and most numerous on the face, chest, and upper back. Normally,
desquamated follicular cells are carried to the surface by the flow of
sebum. Under the abnormal circumstances of acne vulgaris, an abnormal
desquamination process provokes increased sloughing of the epithelium,
which becomes more cohesive because of defective keratinization. This
process causes blockage of the follicular orifice with accumulation of
dead cells. Androgen stimulates the undifferentiated hormonally
responsive cells making up the outer layer of the sebaceous gland lobule
to divide and differentiate. Sebum production favors proliferation of the
anaerobe Propionibacterium acnes, which is a normal commensal to the
pilosebaceous unit, which can elicit hypersensitivity responses in acne.

[0093]The aims of treating acne are to minimize the number and severity of
lesions, prevent scarring, limit disease duration, and reduce the social
and psychological stress that affects many patients, particularly
teenagers. Conventional treatment is directed at correcting the three
major factors that seem to cause acne: (1) androgenic stimulation of the
sebaceous glands and increased sebum production; (2) abnormal
keratinization and impaction in the pilosebaceous canal causing
obstruction to sebum flow; and (3) proliferation of P. acnes. Thus,
topical agents that remove comedones, such as topical retinoids are
particularly effective because they normalize desquamination within the
follicular orifice, which allows the sebum to flow freely onto the
surface of the skin; adalpalene, tretinoin, and tazarotene have been
shown to have efficacy in treating mild to moderate acne, but all three
have reported to have skin-irritating side effects including erythema,
pruritis, burning/stinging, and scaling/flaking (Physicians'Desk
Reference®, 56th ed. 2002, p. 2523, hereinafter referred to as
"PDR"). The side effects of retinoid use are so extreme that many
individuals cannot tolerate topical application of these agents at all.

[0094]The nitroalkene compositions and methods of the present invention
are expected to be effective to prevent and to treat acne. After
treatment for a period of time, decreased inflammation, irritation and
erythema of the skin. This should result in an elimination of acne and
repair microscarring of the dermis from prior acne lesions.

[0095]Psoriasis is another inflammatory skin disease that occurs when
faulty signals in the immune system cause keratinocyte skin cells to
regenerate too quickly, on the order of every three to four days instead
of the usual 30-day cycle. Extra skin cells build up on the skin's
surface, forming red, flaky, scaly lesions that can itch, crack, bleed
and be extremely painful. Psoriasis generally involves the joints, limbs
and scalp but it can appear anywhere on the body, covering some people
from head to toe. More than 5 million Americans have been diagnosed with
psoriasis and/or psoriatic arthritis, a degenerative disease of the
joints and connective tissues associated with psoriasis. Psoriasis
typically first strikes people between the ages of 15 and 35, but can
affect anyone at any age, including children.

[0096]Psoriasis is characterized by erythematous eruptions, often in
papules or plaques, and usually having a white, silvery scale. Numerous
studies have identified tumor necrosis factor α(TNF-α) as a
particularly relevant cytokine regulating this complex inflammatory
cascade. Its key role is underlined by the therapeutic efficacy of
compounds that interfere with TNF-α, functions. It is thought that
neutrophils, another leukocyte population abundantly present in psoriatic
infiltrates, are recruited by the neutrophil-attracting chemokine
interleukin-8 (CXCL8). However, this pathway is probably not the
exclusive means of neutrophil recruitment.

[0097]The nitroalkene compositions and methods of the present invention
are expected to be effective to prevent and to treat psoriasis.

[0098]The present invention thus prevents skin aging and treats skin
aging, as well as both preventing and treating skin damage including
inflammation, scarring and erythema.

[0099]The above description is for the purpose of teaching the person of
ordinary skill in the art how to practice the present invention, and it
is not intended to detail all those obvious modifications and variations
of it which will become apparent to the skilled worker upon reading the
description. It is intended, however, that all such obvious modifications
and variations be included within the scope of the present invention,
which is defined by the following claims. The claims are intended to
cover the claimed components and steps in any sequence which is effective
to meet the objectives there intended, unless the context specifically
indicates the contrary.